Dušanka Milojković-Opsenica

The determination of phenolic profiles of Serbian unifloral honeys using ultra-high-performance liquid chromatography/high resolution accurate mass spectrometry

Polyphenolic profiles of 44 unifloral Serbian honeys were analyzed using ultra-high-performance liquid chromatography (UHPLC) coupled with hybrid mass spectrometer which combines the Linear Trap Quadrupole (LTQ) and OrbiTrap mass analyzer. Rapid UHPLC method was developed in combination with a high sensitivity accurate mass scan and a simultaneous data dependent scan. The honey samples were of different botanical origin: acacia (Robinia pseudoacacia), sunflower (Helianthus annuus), linden (Tilia cordata), basil (Ocimum basilicum), buckwheat (Fagopyrum esculentum), oilseed rape (Brassica napus), and goldenrod (Solidago virgaurea). The presence of 43 compounds, mainly flavonoids, was proven in all honey samples by their characteristic mass spectra and fragmentation pattern. Relatively high amounts of chrysin, pinocembrin and galangin were identified in all honey extracts. p-Coumaric acid was not detected in basil, buckwheat and goldenrod honey extracts. A larger amount of gallic acid (max value 1.45 mg/kg) was found in the sunflower honey, while a larger amount of apigenin (0.97 mg/kg) was determined in the buckwheat honey in comparison with other honeys. The samples were classified according to the botanical origin using pattern recognition technique, Principal Component Analysis (PCA). The LTQ OrbiTrap technique was proven to be reliable for the unambiguous detection of phenolic acids, their derivatives, and flavonoid aglycones based on their molecular masses and fragmentation pattern.

Phenolic profile and antioxidant activity of Serbian polyfloral honeys.

A total of 58 polyfloral honey samples from different regions in Serbia were studied to determine their phenolic profile, total phenolic content and antioxidant capacity. UHPLC-LTQ OrbiTrap MS made possible the identification of 36 compounds: 24 flavonoids, two abscisic acids, and 10 phenolic acids and their derivatives. Quantification was done using 14 available standards. Data on phenolics and abscisic acids allowed the discrimination and classification of honeys in accordance to their geographical origin, using pattern recognition techniques, principal component analysis and partial least squares discriminant analysis. Samples originated from Vojvodina and Zlatibor region were clearly distinguished from those from the rest of Serbia because of the presence of dicaffeoylquinic acid, ellagic acid, caffeic acid phenethyl ester, and chlorogenic acid, among others. A good correlation (r = 0.865) was observed between total phenolic content and radical-scavenging activity. Total phenolic content ranged from 0.03 to 1.39 mg GAE/g and radical scavenging activity ranged from 1.31% to 25.61%.

Pattern recognition methods and multivariate image analysis in HPTLC fingerprinting of propolis extracts

High‐performance thin‐layer chromatography (HPTLC) combined with image analysis and pattern recognition methods were used for fingerprinting and classification of 52 propolis samples collected from Serbia and one sample from Croatia. Modern thin‐layer chromatography equipment in combination with software for image processing and warping was applied for fingerprinting and data acquisition. The three mostly used chemometric techniques for classification, principal component analysis, cluster analysis and partial least square‐discriminant analysis, in combination with simple and fast HPTLC method for fingerprint analysis of propolis, were performed in order to favor and encourage their use in planar chromatography. HPTLC fingerprint analysis of propolis was for the first time performed on amino silica plates. All studied propolis samples have been classified in two major types, orange and blue, supporting the idea of existence of two types of European propolis. Signals at specific RF values responsible for classification of studied extracts have also been isolated and underlying compounds targeted for further investigation. Copyright

Ultrahigh‐performance Liquid Chromatography and Mass Spectrometry (UHPLC–LTQ/Orbitrap/MS/MS) Study of Phenolic Profile of Serbian Poplar Type Propolis

INTRODUCTION Propolis is a resinous natural substance collected by honeybees from different plant sources. Due to the presence of various phytochemicals, this bee-product exhibits numerous biological activities, including anti-bacterial, anti-viral, anti-inflammatory, anti-oxidant, immunostimulating and anti-tumour effects. As the chemical composition and biological activity of propolis depend on its botanical and geographical origin, searching for new bioactive substances in various types of propolis from unexplored regions is of great importance. OBJECTIVE The aim of this study is the evaluation of the phenolic profile of poplar propolis samples in order to characterise Serbian propolis, to identify possible new constituents and to specify the phenolic components relevant for differentiation of poplar propolis samples into two subgroups through simultaneous analysis of poplar bud extracts. METHODS Ethanolic extracts of propolis and poplar buds were comprehensively analysed using ultrahigh-performance liquid chromatography coupled with hybrid mass spectrometry, which combines the linear trap quadrupole and Orbitrap MS/MS mass analyser together with chemometric methods. RESULTS Extensive fingerprint analysis of Serbian propolis was achieved for the first time. Seventy-five phenolic compounds were detected. Eight of them were identified in propolis for the first time. Pattern-recognition methods applied to the content of ten quantified phenolics verified the existence of two subgroups of propolis, with galangin, chrysin and pinocembrin as the most influential distinguishing factors. CONCLUSION The phenolic composition of the analysed propolis samples confirm their affiliation to the European poplar type propolis and the existence of two subgroups according to botanical origin.

Chemical composition of two different extracts of berries harvested in Serbia.

Total phenolic content (TPC), total anthocyanin content (TAC), free and total ellagic acid content, sugars, minerals, and radical-scavenging activity were determined in nine berries harvested in Serbia. More than 30 phenolic compounds were identified; among them, 11 polyphenols and cis,trans-abscisic acid were quantified using UHPLC coupled with an LTQ-Orbitrap XL mass analyzer. For the first time chrysin, naringenin, pinocembrin, and galangin were quantified in some of the investigated berry species. The extraction efficiency of the two extraction systems, methanol and acetone, was investigated. It was found that acetone is a better extracting solvent for TPC, whereas more TAC was extracted by methanol. TPC in acetone extracts ranged from 177.51 to 459.71 mg gallic acid equiv/100 g frozen weight. TAC ranged from 5.39 to 96.94 mg cyanidin-3-glucoside/100 g frozen weight in methanol extracts. The amounts of both free and total ellagic acid were found to be higher in the acetone extract in comparison to the methanol extract.

Salting-out thin-layer chromatography of some angiotensin converting enzyme inhibitors on different sorbents

SummaryChromatographic behaviour of six antihypertensive drugs from the class ofACE inhibitors was studied under conditions of salting-out thin-layer chromatography on silica-gel, cellulose and polyacrylonitrile (PANS) as sorbents. For this purpose, different concentrations of aqueous ammonium sulphate solutions were used as solvents. In all cases studied, it was established that hRF values decrease with increasing salt concentrations in the chromatographic solvent systems used. In addition, a linear relationship of the correspondingRM values and ammonium sulphate content in the solvent was observed. To explain these results, a separation mechanism based on hydrophobic interaction between the sorbent and sorbent is proposed.

Structure–retention relationship study of arylpiperazines by linear multivariate modeling

A quantitative structure-retention relationship study has been performed to correlate the retention of 33 newly synthesized arylpiperazines with their molecular characteristics, using thin-layer chromatography. Principal component analysis followed by multiple linear regression (MLR), principal component regression (PCR) and partial least squares (PLS) was performed to identify the most important factors, to quantify their influences, and to select descriptors that best describe the behavior of the compounds investigated. The best statistical performance was achieved by applying PLS regression, leading to the lowest value of the standard error (root mean square errors of calibration of 0.159 and cross-validated value RMSE cross-validation=0.231 units), followed by the PCR (root mean square errors of calibration=0.195 and RMSE cross-validation=0.305) and MLR (R(adj)(2)=0.9499, F=102.017, mean square error=0.052 and predicted residual error sum of squares=2.23). Two factors of the highest influence: surface tension and hydrophilic-lipophilic balance appear as the part of obtained models. In addition, polar surface area and hydrophilic surface area are included by both PLS and PCR models. Moreover, logP has been added to the PLS model. Besides, PCR model includes following descriptors: hydrogen bond acceptor, hydrogen bond donor and LUMO energy, whereas topological descriptors: connectivity indices 0 and 2, and valence index 3 are included in the MLR model.

Salting-out thin-layer chromatography of some macrolide antibiotics

The chromatographic behavior of roxithromycin, midecamycin, erythromycin, azithromycin, and erythromycin ethylsuccinate has been studied by salting-out thin-layer chromatography (SOTLC) with cellulose as a adsorbent and aqueous ammonium sulfate solutions of different concentration as mobile phases. Hydrophobicity was determined from the linear relationships between solute RM values and the ammonium sulfate content of the mobile phase. Hydrophobicity values RM0 and C0 were calculated on the basis of the results obtained. Lipophilicity determined in this way was correlated with calculated log P values.

Examination of the hydrophobicity of ACE inhibitors and their active metabolites by salting-out thin-layer chromatography

The behavior of five ACE inhibitors and their active degradation products in salting-out thin-layer chromatography (SO TLC) has been examined on silica gel, cellulose, and polyacrylonitrile (PAN) with aqueous ammonium sulfate solutions of different concentration as mobile phases. It was found that increasing the concentration of the salt in the mobile phase led to increased RM values for all the substances. Linear relationships were established between salt concentration and RM values and regression data of the plots obtained were used to determine the lipophilicity RM0 and C0. Lipophilicity determined in this way was correlated with calculated log P values. For comparison of these results with chromatographic behavior in conventional RP TLC, the examined substances were subjected to thin-layer chromatography on RP-18 silica gel with methanol—water mobile phases.

Amino acids profile of Serbian unifloral honeys

BACKGROUND The free amino acids profile of 192 samples of seven different floral types of Serbian honey (acacia, linden, sunflower, rape, basil, giant goldenrod, and buckwheat) from six different regions was analysed in order to distinguish honeys by their botanical origin. RESULTS The most abundant amino acids were proline, alanine, phenylalanine, threonine and arginine. Based on the established amino acids profiles, some important differences have been identified among studied honey samples relying on the basic descriptive statistics data, and confirmed by multivariate chemometric methods. Principal component analysis revealed that basil honey samples form a well-defined cluster imposed with phenylalanine content. The model obtained by linear discriminant analysis might be used to distinguish basil honey from the rest of the samples, and has moderate predictive power to separate genuine acacia, linden, sunflower and rape honeys. New data for the amino acids profile of giant goldenrod and buckwheat honey samples are presented. CONCLUSIONS The floral origin of honey could be successfully evaluated by its amino acids profile coupled with chemometric analysis.